Method for detection of a genetic variant

ABSTRACT

A method and kit for detecting a genetic variant associated with a disease or disorder, including incompatibility with a pharmaceutical. The method and kit using a first nano-particle coupled to at least one morpholino nucleic acid probe comprising a target complimentary region base sequence that is a perfect match to a genetic variant sequence.

FIELD OF THE INVENTION

The present invention relates to the detection of genetic variantsincluding methods and kits for detecting the same.

BACKGROUND OF THE INVENTION

Genetic variation among individuals within a population is oftenobserved by phenotypic changes that in some cases manifest as a diseaseor disorder. There are some instances where a disease or disorder onlymanifests as an adverse reaction to an external stimulus such as apharmaceutical drug or other chemicals. The association studies ofadverse drug reactions (ADRs) and patients' genetic makeup hold thepromise of greatly reducing the ADR-related morbidity and mortality bygenetic testing. There are several known links between ADR and geneticvariants.

Fluorouracil (5-FU) has been used as a chemotherapy agent in thetreatment of patients with breast, colorectal, lung and other cancersfor several decades. Side-effects of 5-FU include diarrhea, stomatitis,mucositis, neurotoxicity, and in some cases, death. These are largelydue to genetic inability to metabolize the drug. Dihydropyrimidinedehydrogenase (DPD), an enzyme encoded by the DPD gene, is responsiblefor the elimination of ˜80% of the standard dose of 5-FU. DPD deficiencydue to DPD gene mutations has been associated with severe 5-FU toxicity.It has been found that ˜3-5% of patients harbor at least a partial DPDdeficiency. The most common mutation associated with DPD deficiency isIVS14+1 G>A, a G>A base change at the splice recognition sequence ofintron 14 (known as DPD*2A). This single-nucleotide variation leads toexon skipping and results in a 165-bp deletion in the DPD mRNA. Ahomozygote DPD*2A genotype results in complete deficiency, while theheterozygous DPD*2A genotype results in partial deficiency of DPD. Thepre-therapeutic detection of this metabolic dysfunction could preventsevere side-effects of 5-FU by administering alternative treatments.

Similarly, the linkage of CYP2C19 genotype with clinical outcomes amongclopidogrel-treated acute coronary syndrome (ACS) patients, particularlythose undergoing percutaneous coronary intervention (PCI) has beenreported. Clopidogrel is a commonly prescribed oral, antiplatelet agentused to inhibit blood clots in coronary artery disease, peripheralvascular disease, and cerebrovascular disease. As a thienopyridineprodrug, clopidogrel requires hepatic biotransformation to form anactive metabolite. The hepatic CYP2C19 enzyme is one of CYP450superfamily members that is involved in the metabolism of clopidogrel.The CYP2C19 gene is highly polymorphic with over 25 known variantalleles. The most common CYP2C19 loss-of-function allele is CYP2C19*2(c.681G>A; rs4244285), with allele frequencies of ˜15% in Caucasians andAfricans, and 29-35% in Asians. However, current genetic testing methodsfor loss of function generally require expensive instruments and longturnaround time.

Another example is the discovery of the strong linkage betweencarbamazepine-induced Steven Johnson Syndrome/Toxic Epidermal Necrolysis(SJS/TEN) and a specific allele of human leukocyte antigen (HLA),HLA-B*1502. Carbamazepine is the first-line drug for treatment ofpatients with epilepsy, neuropathic pain and bipolar disorder. However,carbamazepine therapy may cause dangerous or even fatal skin reactions,such as SJS and TEN in patients with the HLA-B*1502 allelic variation.Prevalence of the HLA-B*1502 allele in Asian populations, except inJapanese and Koreans, is much higher than in Caucasian and Africanpopulations. Some studies revealed that the negative predictive value ofHLA-B*1502 test for carbamazepine-induced SJS could be close to 100%.HLA-B gene is highly polymorphic with more than 2000 unique alleles,which complicates the determination of a specific allele. Sangersequencing is considered to be the gold standard for HLA typing.However, the phase ambiguity often causes problems for SBT inidentification of allele combinations. Next-generation sequencing (NGS)technologies are able to obtain the sequence of a single DNA moleculeand may rule out the phase ambiguity, but drawbacks of the current NGSmethods include the short read length, high workloads for librarypreparation and lengthy processing time. A recent report employedloop-mediated isothermal amplification (LAMP) for HLA-B*1502 screening(Cheng, et al. Clin. Chem. 2009, 55, 1568.). Unfortunately, thespecificity of these methods is not sufficient to distinguish HLA-B*1502from quite a few other alleles that are not rare in some populations.The false assays will rule out the patients with very low SJS risk fromthe effective carbamazepine therapy, and increase the treatment cost.Since the cost-effectiveness of HLA-B*1502 screening is highly dependenton the accuracy and cost of the genetic test, there is an urgent needfor a new assay that is more specific and less costly.

The US Food and Drug Administration (FDA) and other regulatory bodieshave required labelling of several drugs that may have adverse effectsin certain genotypes. For Example a requirement for fluorouracil(5-FU)-based drugs is to contain a warning for hypersensitivity in someindividuals. In another example, the US Food and Drug Administrationissued an alert in 2007 that recommends screening of the HLA-B*1502allele before starting treatment with carbamazepine for patients withancestry from areas in which the specific allele is present. Similarly,the boxed warning on the clopidogrel label recommends consideration ofalternative antiplatelet therapy in poor metabolizers with ACS or PCI.

Commercial kits for genotyping tests are available for some geneticvariants and are used to predict an individual's tolerance orintolerance of a drug. The testing platforms used include Sangersequencing and restriction fragment length polymorphism (RFLP) analysis.The assays based on these platforms are either time-consuming or costly,and therefore, less suitable for on-demand testing. Commercial kit basedon real-time PCR is also available unfortunately, the specificity ofthese methods is not sufficient to distinguish complex allelic variantsfrom quite a few other alleles that are not rare in some populations.Since the cost-effectiveness of screening is highly dependent on theaccuracy and cost of the genetic test, there is an urgent need for a newassay that is more specific and less costly.

SUMMARY OF THE INVENTION

The present invention meets this need by providing, in a first aspect ofthe invention, a method of detecting a genetic variant associated with adisease or disorder, including incompatibility with a pharmaceutical,the method comprising the steps of

a) providing a first nano-particle coupled to at least one morpholinonucleic acid probe comprising a target-complementary region comprising abase sequence that is a perfect match to the genetic variant;

b) combining a sample containing nucleic acid suspected of containingthe genetic variant and the first nano-particle to provide a firstmixture under conditions that allow hybridization of the morpholinonucleic acid probe and the nucleic acid suspected of containing thegenetic variant;c) sequentially heating the first mixture and determining the meltingtemperature of the hybridization complex between the morpholino nucleicacid probe and the nucleic acid suspected of containing the geneticvariant;d) comparing the melting temperature determined in step c) with astandard to determine whether the sample comprises nucleic acidcontaining the genetic variant.

Another aspect of the invention relates to a detection kit forperforming the methods described herein, comprising: a firstnano-particle coupled to at least one morpholino nucleic acid probecomprising a target complimentary region comprising a base sequence thatis a perfect match to a genetic variant sequence associated with adisease or disorder, including incompatibility with a pharmaceutical.

Other aspects of the invention will be apparent to a person skilled inthe art with reference to the following drawings and description ofvarious non-limiting embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not necessarily drawn to scale, emphasis insteadgenerally being placed upon illustrating the principles of variousembodiments. In the following description, various embodiments of theinvention are described with reference to the following drawings.

FIG. 1. Sequences of the MORs used in the DPD tests. The single-basedifferences for each pair of the probes are underlined. FIG. 1corresponds to the following SEQ ID Nos.: SEQ ID Nos. 1 and 2.

FIG. 2. Sequences of the synthetic DNA samples used in the DPDgenotyping analysis. The single-nucleotide polymorphism (SNP) positionsare underlined. FIG. 2 corresponds to the following SEQ ID Nos.: SEQ IDNo. 19.

FIG. 3. PCR primers for DPD target sequence amplification. FIG. 3corresponds to the following SEQ ID Nos.: SEQ ID Nos. 9 and 10.

FIG. 4. Thermal cycler protocol for PCR amplification of DPD sequences.

FIG. 5. Melting temperature as a function of DPD target concentrationfor the (a) WT and (b) MUT probes targeting the DPD*2A SNP. Samples aresynthetic single-stranded wild-type DNA (circle), mutant DNA (square),and their 1:1 mixture (triangle, representing heterozygote samples).Error of T_(m) measurement=±1° C.

FIG. 6. Scatter plots of T_(m) ^(WT) and T_(m) ^(MUT) of the DPDsynthetic DNA samples. Error of T_(m) measurement=±1° C.

FIG. 7. Alignment of typical HLA-B alleles. FIG. 7 corresponds to thefollowing SEQ ID Nos.: SEQ ID Nos. 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 43.

FIG. 8. PCR primer and probe design for HLA-B*1502 allele determination.

FIG. 9. Combination ambiguities in HLA-B alleles of PCR1.

FIG. 10. Sequences of the MORs used in the HLA-B tests. FIG. 10corresponds to the following SEQ ID Nos.: SEQ ID Nos. 3, 4, 5 and 6.

FIG. 11. Sequences of the synthetic DNA samples used in the HLA-Bgenotyping analysis. The nucleotide variants are highlighted byunderlining. FIG. 11 corresponds to the following SEQ ID Nos.: SEQ IDNos. 44, 45, 46, 47, 48, 49, 50 and 51.

FIG. 12. Assay workflow of the HLA-B tests.

FIG. 13. Sequences of PCR primers used in the HLA-B tests. FIG. 13corresponds to the following SEQ ID Nos.: SEQ ID Nos. 11, 12, 13, 14, 17and 18.

FIG. 14. Thermal cycler protocol for PCR amplification of HLA-Bsequences.

FIG. 15. Melting temperature as a function of target concentration fornanoprobes 1, 2 and 3 for the HLA-B tests.

FIG. 16. Sequences of the MORs used in the CYP2C19 tests. Thesingle-base difference is highlighted by underlining. FIG. 16corresponds to the following SEQ ID Nos.: SEQ ID Nos. 7 and 8.

FIG. 17. Melting temperature as a function of target concentration forthe (a) WT and (b) MUT probes targeting the CYP2C19*2 SNP. Samples aresynthetic single-stranded wild-type DNA (circle), mutant DNA (square),and their 1:1 mixture (triangle, representing heterozygote samples).Error of T_(m) measurement=±1° C.

FIG. 18. Sequences of the CYP2C19 synthetic DNA samples used in thiswork. The SNP position is highlighted by underlining. FIG. 18corresponds to the following SEQ ID Nos.: SEQ ID No. 52.

FIG. 19. Scatter plots of T_(m) ^(WT) and T_(m) ^(MUT) of the CYP2C19synthetic DNA samples. Error of T_(m) measurement=±1° C.

FIG. 20. PCR primers for CYP2C19 target sequence amplification.

FIG. 21. Thermal cycler protocol for CYP2C19 PCR amplification.

DETAILED DESCRIPTION

The inventors have developed a cost-effective genotyping platform usingnanoprobes coupled to specific morpholino oligo sequences. Thecorresponding assay kit can greatly promote the translation of thepharmacogenomic knowledge in clinical practice.

A first aspect of the invention relates to a method of detecting agenetic variant associated with a disease or disorder, includingincompatibility with a pharmaceutical, the method comprising the stepsof

a) providing a first nano-particle coupled to at least one morpholinonucleic acid probe comprising a target-complementary region comprising abase sequence that is a perfect match to the genetic variant;

b) combining a sample containing nucleic acid suspected of containingthe genetic variant and the first nano-particle to provide a firstmixture under conditions that allow hybridization of the morpholinonucleic acid probe and the nucleic acid suspected of containing thegenetic variant;c) sequentially heating the first mixture and determining the meltingtemperature of the hybridization complex between the morpholino nucleicacid probe and the nucleic acid suspected of containing the geneticvariant;d) comparing the melting temperature determined in step c) with astandard to determine whether the sample comprises nucleic acidcontaining the genetic variant.

The term “genetic variant” as used herein refers to a naturallyoccurring gene sequence that deviates from a predominant genotype of apopulation resulting in a variation of a phenotype associated with adisease or disorder, including incompatibility with a pharmaceuticaldrug. The predominant genotype may be defined to be that sequence whichis present in a majority of the population or the most frequentgenotype. In various embodiments, the predominant genotype comprises thesame gene sequence in at least 55% of the population, or at least 60%,or at least 70%, or at least 80%, or at least 90%, or at least 95% ofthe population. Herein, the term “predominant genotype” is also referredto as the “wildtype”. Accordingly, the wildtype or predominant sequencedoes not comprise the genetic variation that is responsible for thedisease or disorder. Examples of genetic variants include deletions ofnucleic acids, addition of nucleic acids, single nucleotide polymorphism(SNP) or allelic variants. The term “genetic variant”, as used herein,thus means a specific variation in the base sequence of a given gene.

The morpholino nucleic acids of the present invention arephosphorodiamidate morpholino oligonucleotides (PMO), wherein the sugarand phosphate backbone is replaced by morpholine groups linked byphosphoramidates, and the nucleobases, such as cytosine, guanine,adenine, thymine and uracil, preferably adenine, guanine, cytosine andthymine, are coupled to the morpholine ring. The PMOs typically comprisemonomeric units of the structure:

The terms “morpholino”, “morpholino nucleic acid” and “morpholinooligo(nucleotide)” are used interchangeably herein and refer to theafore-mentioned phosphorodiamidate morpholino oligonucleotides (PMOs).

In various embodiments the morpholino oligo is covalently coupled to thenanoparticle. This may for example be achieved via the 5′-terminalphosphoramidate group or the 3′-terminal ring nitrogen. The length ofthe morpholino oligonucleotides described herein can comprise about 5monomeric units to about 40 monomeric units; about 10 monomeric units toabout 35 monomeric units; or about 15 monomeric units to about 35monomeric units.

As used herein, the term “complementary” or “complementarity” relates tothe relationship of nucleotides/bases on two different strands of DNA orRNA, or the relationship of nucleotides/bases of the base sequence ofthe morpholino and a DNA/RNA strand, where the bases are paired byWatson-Crick base pairing, i.e. guanine with cytosine, adenine withthymine (DNA) or uracil (RNA). Accordingly, the morpholino as describedherein comprises a base sequence that can form hydrogen bond(s) with atarget nucleotide sequence, for example a DNA or RNA sequence, byconventional Watson-Crick base pairing between complementary bases.

In this context, the term “hybridize” or “hybridization” refers to theinteraction between two different strands of DNA or RNA or betweennucleotides/bases of the base sequence of the morpholino and a DNA/RNAsequence by hydrogen bonds in accordance with the rules of Watson-CrickDNA complementarity.

A target-complementary region is a base sequence that hascomplementarity to a given target sequence and hybridizes to said targetunder conditions that allow such hybridization. “Perfectly matched”, asused in this connection, refers to the feature that a base sequence is100% complementary to the target nucleic acid sequence, i.e. all basesof the given sequence, for example the target-complementary region, formWatson-Crick base pairs with a contiguous nucleotide sequence in thetarget.

The term “nanoparticle” as used herein refers to any particle having asize from about 1 to about 250 nm as long as the nanoparticle is capableof providing optical properties; for example, generate optical signalssensitive to hybridization reactions. The diameter of the nanoparticleas described herein can range in the size from about 1 nm to about 250nm; about 1 nm to about 200 nm; about 1 nm to about 160 nm; about 1 nmto about 140 nm; about 1 nm to about 120 nm; about 1 nm to about 80 nm;about 1 nm to about 60 nm; about 1 nm to about 50 nm; about 5 nm toabout 250 nm; about 8 nm to about 250 nm; about 10 nm to about 250 nm;about 20 nm to about 250 nm; about 30 nm to about 250 nm; about 40 nm toabout 250 nm; about 85 nm to about 250 nm; about 100 nm to about 250 nm;or about 150 nm to about 250 nm. In some embodiments, the diameter ofthe diameter of the nanoparticle is in the range of about 1 nm to about100 nm.

In certain embodiments, the nanoparticle is a metal nanoparticle. Inother embodiments, the nanoparticle is a colloidal metal.

In some embodiments, the metal is a noble metal. Non-limiting examplesof a noble metal that can be used can include silver, gold, platinum,palladium, ruthenium, osmium, iridium or mixtures thereof, not tomention a few. Other metals that can also be used in the formation ofthe nanoparticle can include but are not limited to aluminium, copper,cobalt, indium, nickel, or any other metal amenable to nanoparticleformation). The nanoparticle as described herein can also comprise asemiconductor (including for example and without limitation, CdSe, CdS,and CdS or CdSe coated with ZnS) or magnetic (for example,ferromagnetite) colloidal materials. Other nanoparticles useful in thepractice of the invention include, also without limitation, ZnS, ZnO,Ti, TiO₂, Sn, SnO₂, Si, SiO₂, Fe, Ag, Cu, Ni, Al, steel, cobalt-chromealloys, Cd, titanium alloys, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe,In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs.

Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe,CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticles arealso known in the art. See for e.g., Weller, Angew. Chem. Int. Ed.Engl., 32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988).Methods for making metal, semiconductor and magnetic nanoparticles arealso well-known in the art, see for example, Ahmadi, T. E. et al.,Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99,14129 (1995). Suitable nanoparticles are also commercially availablefrom, for example, Ted Pella, Inc. (gold), Amersham Corporation (gold)and Nanoprobes Inc (gold). The nanoparticles comprising materialsdescribed herein are available commercially or they can be produced fromprogressive nucleation in solution (e.g., by colloid reaction), or byvarious physical and chemical vapour deposition process, such as sputterdisposition. The nanoparticles as described herein can also be producedusing HAuCl₄ and a citrate-reducing agent, using methods known in theart (see for example, Grabar, K. C. et al, Anal. Chem., 1995, 67,735-743).

The morpholino oligo can be coupled to the nanoparticle by any suitablemeans. The coupling is preferably covalent coupling. It is furtherpreferred that each nanoparticles comprises at least one, preferablymore than one probe coupled to its surface. The coupling may for examplebe done by functionalizing the oligo with a group that can react with agiven nanoparticle. In case the nanoparticle is a gold nanoparticle, thefunctional group can, without limitation, be a sulfur-containing group,such as a sulfide or thiol group.

The combining step may be done by combining the sample with thenanoparticles, preferably in form of a dispersion in an aqueous medium,under conditions that allow hybridization of the probe coupled to thenanoparticle to the target nucleic acid. These conditions may includethe use of suitable buffers and temperature conditions, for exampleheating to a temperature that dissociates existing double-strandedstructures and cooling to allow hybrid formation. The conditions may bestrict to avoid hybridization with non-target nucleic acids. Typicalconditions, including buffers and temperature conditions, forprobe-target hybridization are well-known in the art.

The sample may be any suitable sample, but is preferably a biologicalsample that contains nucleic acid molecules. Such samples may, forexample, be derived from tissues and body fluids or any other biologicalsamples that contain cells or cell components. The term “body fluid”, asused in this connection, includes blood, plasma, serum, lymph fluid andthe like. Typically, the biological sample is a sample that containsintact cells, such as a mouth swap. The sample may be processed beforebeing used in the described methods, for example subjected to variousisolation/purification steps, all of which are known to those skilled inthe art.

Melting temperature as used herein is the temperature at which a complexof the morpholino oligo and a nucleic acid dissociate into singlestranded state. Thermal denaturation referred to as melting, comprisesheating double stranded duplexes to break the hydrogen bonds formedbetween bases after which the two strands separate. Due to differentmolecular geometrics of nucleotides, a single inconsistency between thetwo strands will make binding between them less energeticallyfavourable. This will result in perfect matched bases melting at ahigher temperature than mismatched strands. A morpholino oligo with aperfect match to the genetic variant is typically designed such that itwill however hybridize to both the genetic variant and the wildtypesequences, but the temperature at which the morpholino oligo dissociatesfrom the genetic variant nucleic acid sequence will be higher than thetemperature at which the morpholino oligo dissociates from the wildtypenucleic acid sequence. Similarly, where a morpholino oligo is a perfectmatch to the wildtype sequence it will hybridise to both the geneticvariant and the wildtype sequences, but the temperature at which themorpholino oligo dissociates from the genetic variant nucleic acidsequence will be lower than the temperature at which the morpholinooligo dissociates from the wildtype nucleic acid sequence.

To determine the melting temperature, the hybridized complexes that formbetween the morpholino oligo probes and the nucleic acids aresequentially heated, for example by about one degree (° C.) in a giventime interval. The melting temperature (T_(m)) is measured by meansknown in the art, for example change in absorbance intensity such aschange in UV absorbance, alternatively by using intercalatingfluorophores and measuring changes in fluorescence. In variousembodiments, the melting temperature is indicated by a color change ofthe mixture upon dissociation of the probe and the sample nucleic acid.In these embodiments, because the signals can be visualized by the nakedeye and/or recorded by a digital camera, no expensive detector isrequired. Thus, the method allows for rapid, convenient andcost-effective genetic testing.

Nobel metals exhibit variations in plasmon resonance due to changes inthe local index of refraction. As small nanoparticles in solution, noblenanoparticles will appear as one colour and if they aggregate to form alarger cluster a colour change can be observed. In various embodimentsgold nanoparticles are used together with the morpholino oligo probewhereby when the morpholino oligo is hybridised to a nucleic acid, thesolution appears red and when the morpholino oligo is dissociated fromthe nucleic acid at the melting temperature, the nanoparticles aggregateand the solution appears grey. In other words the colour change iscaused by the probe-target hybridization preventing aggregation of theparticles, for example by sterically preventing the interaction betweenthe individual particles, but once the target is dissociated, theinteraction between the individual particles is no longer prevented andthey aggregate. To achieve this switching between the non-aggregatedstate and the aggregated state, the nanoparticles comprising probeshybridized to the target may be subjected to conditions that wouldnormally lead to particles aggregation but are insufficient to causeaggregation of the particles complexed with the target. Such conditionsmay, for example and without limitation, include buffer conditions withincreased ionic strength, for example by high salt concentrations.Normally, such increased salt conditions lower the hydration of thenanoparticles and thus promote aggregation. However, in case theparticles are complexed with the target the increase in ion strength isnot sufficient to lead to aggregation so that aggregation only occursonce the nanoparticles are dissociated from the target nucleic acid.

Once the melting temperature has been determined, the obtained value iscompared with a standard value which allows determination of thepresence of the genetic variant. Depending on the type of standard,either an increase of the determined melting temperature compared to thestandard or an equal value can indicate the presence of the geneticvariant. If the presence of the genetic variant is indicated by anincrease in melting temperature, the increase is preferably large enoughto make specific detection possible. For example, the increase inmelting temperature can be at least about 0.5° C., preferably at leastabout 1° C.

In various embodiments, the standard is established by determining themelting temperature of a complex of the first nano-particle coupled toat least one morpholino nucleic acid probe comprising atarget-complementary region comprising a base sequence that is a perfectmatch to the genetic variant and the corresponding wildtype nucleicacid, wherein when the comparison in step d) shows that the meltingtemperature determined in step c) is higher than the melting temperatureof the complex of the first nano-particle and the wildtype nucleic acid,this is indicative that the sample comprises nucleic acid containing thegenetic variant.

In other various embodiments, the standard is established by determiningthe melting temperature of a complex of a second nano-particle coupledto at least one morpholino nucleic acid probe comprising atarget-complementary region comprising a base sequence that is a perfectmatch to the corresponding wildtype base sequence and the wildtypenucleic acid, wherein when the comparison in step d) shows that themelting temperature determined in step c) is equal to the meltingtemperature of the complex of the second nano-particle and the wildtypenucleic acid, this is indicative that the sample comprises nucleic acidcontaining the genetic variant.

In still further embodiments, the standard is established by determiningthe melting temperature of a complex of a second nano-particle coupledto at least one morpholino nucleic acid probe comprising atarget-complementary region comprising a base sequence that is a perfectmatch to the corresponding wildtype base sequence with the nucleic acidcontaining the genetic variant, wherein when the comparison in step d)shows that the melting temperature determined in step c) is higher thanthe melting temperature of the complex of the second nano-particle andthe nucleic acid containing the genetic variant, this is indicative thatthe sample comprises nucleic acid containing the genetic variant;

In various embodiments, the standard is established by using syntheticsequences for either the genetic variant nucleic acid or the wildtypenucleic acid sequence.

In various embodiments, the method further comprises the steps ofestablishing a standard by

a1) providing a second nano-particle coupled to at least one morpholinonucleic acid probe comprising a target-complementary region comprising abase sequence that is a perfect match to the corresponding wildtype basesequence of the genetic variant;

b1) combining a sample containing nucleic acid suspected of containingthe genetic variant and the second nano-particle to provide a secondmixture under conditions that allow hybridization of the morpholinonucleic acid probe and the nucleic acid suspected of containing thegenetic variant; andc1) sequentially heating the second mixture and determining the meltingtemperature of the hybridization complex between the morpholino nucleicacid probe and the nucleic acid suspected of containing the geneticvariant; andwherein step d comprises comparing the melting temperature determined instep c) with the melting temperature determined in step c1), whereinwhen the melting temperature determined in step c) is higher than thestandard melting temperature determined in step c1), this is indicativethat the sample comprises nucleic acid containing the genetic variant.

The detection is comparative and dependant on the most specifichybridization, such that when the genetic variant sequence is present ina sample taken from an individual then the perfectly complimentarymorpholino base sequence of the first probe will have a more specifichybridization than a morpholino base sequence of the second probeperfectly complimentary to the wildtype sequence and will result inhigher melting temperatures for the first probe. In contrast, when thegenetic variant sequence is not present in a sample taken from anindividual and the wildtype genetic sequence is present then theperfectly complimentary morpholino base sequence of the first probe willhave a less specific hybridization than a morpholino base sequence ofthe second probe perfectly complimentary to the wildtype sequence andwill result in lower melting temperatures for the first probe.

In various embodiments the genetic variant is associated with a SNP ofthe gene encoding dihydropyrimidine dehydrogenase (DPD), said SNPresulting in incompatibility with the pharmaceutical compoundFluorouracil.

The SNP of the gene encoding Dihydropyrimidine Dehydrogenase may include*2A (DPD*2A) or [cytochrome P450]2C19*2 genotypes

In this particular embodiment, the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to a sequence related to said SNP of DPD and having thebase sequence set forth in SEQ ID NO:1 (CACTTATGTTGTCTGTTTTTTTTTT) andthe wild type morpholino nucleic acid probe comprises atarget-complementary region being fully complementary to the wild typesequence having the base sequence set forth in SEQ ID NO:2(CACTTACGTTGTCTGTTTTTTTTTT).

The analysis of the data using the DPD sequences leads to unambiguousgenotype assignment.

In various embodiments the genetic variant is associated with a SNP ofthe gene encoding CYP2C19 enzyme, said SNP resulting in incompatibilityof the pharmaceutical compound Clopidogrel.

The allelic variant of the gene encoding CYP2C19 enzyme may includeCYP2C19*2 (c.681G>A; rs4244285).

In this particular embodiment the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to a sequence related to said SNP having the base sequenceset forth SEQ ID NO:7 (GTTATGGGTTCCTGGTTTTTTTTTT) and the wild typemorpholino nucleic acid probe comprises a target-complementary regionbeing fully complementary to the wild type sequence having the basesequence set forth in SEQ ID NO:8 (GTTATGGGTTCCCGGTTTTTTTTTT).

In various embodiments the genetic variant is associated with theallelic variation of the gene encoding human leukocyte antigen, saidallelic variant resulting in incompatibility of the pharmaceuticalcompound Carbamazepine.

The allele variant of the gene encoding human leukocyte antigen mayincludes Human Leukocyte Antigen-B*1502 (HLA-B*1502).

In this particular embodiment the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to an allelic variant having the base sequence set forthin SEQ ID NO:3 (GTGTTCCGATCCCAATTTTTTTTTT); SEQ ID NO:4(TGGTCTTGGAGATCTTTTTTTTTTT); and SEQ ID NO:5(AGGTTCCGCAGGCTTTTTTTTTTTT).

In various embodiments the method may optionally comprises a furtherfunctionalized nano-particle comprising a morpholino nucleic acid probecomprising a target-complementary region being fully complementary to ahousekeeping gene. In one such embodiment the housekeeping gene is GADPHhaving a morpholino sequence set forth in SEQ ID NO:6(CAAGCTTCCCGTTCTCAGCCTTTTT). Such nanoparticle with a probe for ahousekeeping gene may function as a positive control.

In various embodiments the method further comprises enriching and/oramplifying the nucleic acid suspected of containing the genetic variantprior to combining the nucleic acid with the first or the secondnano-particle. The enrichment is preferably done by amplification, morepreferably by using polymerase chain reaction (PCR) to amplify thestretch of the nucleic acid containing the target sequence, i.e. thesequence comprising the genetic variant. This provides the advantagethat the minimum amount needed for detection can be generated in caseswhere the genetic variant exists in low copy number in cells or whereonly small sample volumes are available.

Two separate PCR reactions have been designed to gauge HLA-B*1502 allelein a highly specific manner. The method shows unique features andsuperior performance as compared with other available techniques.Firstly, the assay is highly specific, ensuring accurate detection.Secondly, the nanoprobe technique allows for distinct colorimetricdetection, greatly reducing the screening cost. As a result, thenanoprobe-based genetic testing provides a highly specific and lesscostly assay for the screening of HLA-B*1502.

In various embodiments when the genetic variant is associated with anSNP of the gene encoding dihydropyrimidine dehydrogenase (DPD), thenucleic acid is amplified using a primer pair comprising SEQ ID NO:9(DPD-Forward AGTGAGAAAACGGCTGCATAT) and SEQ ID NO:10 (DPD-ReverseCATTCACCAACTTATGCCAATTCTCTT).

In various embodiments when the genetic variant is associated with theallelic variation of the gene encoding human leukocyte antigen, thenucleic acid is amplified with a primer pair comprising SEQ ID NO:11(P1-Forward CGC GAGTCC GAG GAT GGC) and SEQ ID NO:12 (P1-Reverse CG CAGCCA TAC ATC CTC TGG ATGA) and/or SEQ ID NO:13 (P2-Forward GGA GTA TTGGGA CCG GAAC) and SEQ ID NO:14 (P2-Reverse GTT GTA GTA GCC GCG CAG GT).

In various embodiments when the genetic variant is associated with anSNP of the gene encoding CYP2C19 enzyme, the nucleic acid is amplifiedwith a primer pair comprising SEQ ID NO:15(Forward-TGCAATAATTTTCCCACTATCATTG), and SEQ ID NO:16(Reverse-CTCCAAAATATCACTTTCCATAAAAGCA).

In various embodiments the method further comprising the primer pair SEQID NO:17 (GAPDH-Forward GGAAGGTGAAGGTCGGAGTC CTC) and SEQ ID NO:18(GAPDH-Reverse CCTGGAAGATGGTGATGGGATTTC) for amplifying the housekeepinggene.

Another aspect of the invention relates to a detection kit forperforming the methods described herein comprising; a firstnano-particle coupled to at least one morpholino nucleic acid probecomprising a target complimentary region comprising a base sequence thatis a perfect match to a genetic variant sequence associated with adisease or disorder, including incompatibility with a pharmaceutical.

In various embodiments the kit further comprises a second nano-particlecoupled to at least one morpholino nucleic acid probe comprising atarget complimentary region comprising a base sequence that is a perfectmatch to a wildtype sequence associated with a disease or disorder.

In various embodiments the target-complementary region of the firstprobe is selected from the group consisting of the base sequences setforth in SEQ ID Nos. 1, 3-5 and 7. In case the first probe has the basesequence of SEQ ID NO:1, the target-complementary region of the secondprobe may consist of the base sequence set forth in SEQ ID NO:2. In casethe first probe has the base sequence of SEQ ID NO:7, thetarget-complementary region of the second probe may consist of the basesequence set forth in SEQ ID NO:8.

In various embodiments the kit further comprises a functionalizednano-particle comprising a morpholino nucleic acid probe comprising atarget-complementary region that is fully complementary to ahousekeeping gene.

In various embodiments the housekeeping gene preferably comprises GADPHhaving a morpholino sequence set forth in SEQ ID NO. 6(CAAGCTTCCCGTTCTCAGCCTTTTT).

In various embodiments the nanoparticle and the morpholino nucleic acidsof the kit are as described above in connection with the methods of thepresent invention.

In various embodiments the genetic variant is associated with a SNP ofthe gene encoding dihydropyrimidine dehydrogenase (DPD), said deficiencyresulting in incompatibility with the pharmaceutical compoundFluorouracil.

The SNP of the gene encoding Dihydropyrimidine Dehydrogenase may include*2A (DPD*2A) or [cytochrome P450]2C19*2 genotypes

In this particular embodiment the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to a sequence related to said SNP and having the basesequence set forth in SEQ ID NO:1 (CACTTATGTTGTCTGTTTTTTTTTT) and thewild type morpholino nucleic acid probe comprises a target-complementaryregion being fully complementary to the wild type sequence having thebase sequence set forth in SEQ ID NO:2 (CACTTACGTTGTCTGTTTTTTTTTT).

The genetic information provided by the assay kit can be used to predictDPD deficiency, and consequently, the toxicity of Fluorouracil. Themethod is simple and more cost-effective than other genotypingtechnologies.

In various embodiments the genetic variant is associated with a SNP ofthe gene encoding CYP2C19 enzyme, said SNP resulting in incompatibilityof the pharmaceutical compound Clopidogrel.

The SNP of the gene encoding CYP2C19 enzyme may include CYP2C19*2(c.681G>A; rs4244285).

In this particular embodiment the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to a sequence related to said SNP having the base sequenceset forth in SEQ ID NO:7 (GTTATGGGTTCCTGGTTTTTTTTTT) and the wild typemorpholino nucleic acid probe comprises a target-complementary regionbeing fully complementary to the wild type sequence having the basesequence set forth in SEQ ID NO:8 (GTTATGGGTTCCCGGTTTTTTTTTT).

In various embodiments the genetic variant is associated with an allelicvariation of the gene encoding human leukocyte antigen, said allelicvariant resulting in incompatibility of the pharmaceutical compoundCarbamazepine.

The allele variant of the gene encoding human leukocyte antigen mayincludes Human Leukocyte Antigen-B*1502 (HLA-B*1502).

In this particular embodiment the morpholino nucleic acid probeoptionally comprises a target-complementary region being fullycomplementary to an allelic variant having the base sequence set forthin SEQ ID NO:3 (GTGTTCCGATCCCAATTTTTTTTTT); SEQ ID NO:4(TGGTCTTGGAGATCTTTTTTTTTTT); and SEQ ID NO:5(AGGTTCCGCAGGCTTTTTTTTTTTT).

In various embodiments the kit may further comprise a functionalizednano-particle comprising a morpholino nucleic acid probe being a perfectmatch to a wild type sequence associated with a compatibility of thepharmaceutical.

In various embodiments the kit further comprises a primer pair foramplification of a nucleic acid prior to detection with thenanoparticles.

In various embodiments the primer pair is selected from the groupconsisting of SEQ ID NO:9 (DPD-Forward AGTGAGAAAACGGCTGCATAT) and SEQ IDNO:10 (DPD-Reverse CATTCACCAACTTATGCCAATTCTCTT) SEQ ID NO:11 (P1-ForwardCGC GAGTCC GAG GAT GGC) and SEQ ID NO:12 (P1-Reverse CG CAG CCA TAC ATCCTC TGG ATGA) and/or SEQ ID NO:13 (P2-Forward GGA GTA TTG GGA CCG GAAC)and SEQ ID NO:14 (P2-Reverse GTT GTA GTA GCC GCG CAG GT) SEQ ID NO:15(Forward-TGCAATAATTTTCCCACTATCATTG), and SEQ ID NO:16(Reverse-CTCCAAAATATCACTTTCCATAAAAGCA).

In various embodiments wherein when the genetic variant is associatedwith the SNP of the gene encoding dihydropyrimidine dehydrogenase (DPD),said SNP resulting in incompatibility with the pharmaceutical compoundFluorouracil the primer pair comprises SEQ ID NO:9 (DPD-ForwardAGTGAGAAAACGGCTGCATAT) and SEQ ID NO:10 (DPD-ReverseCATTCACCAACTTATGCCAATTCTCTT).

In various embodiments wherein when the genetic variant is associatedwith the allelic variation of the gene encoding human leukocyte antigen,said allelic variant resulting in incompatibility of the pharmaceuticalcompound Carbamazepine the primer pair comprises SEQ ID NO. 11(P1-Forward CGC GAGTCC GAG GAT GGC) and SEQ ID NO. 12 (P1-Reverse CG CAGCCA TAC ATC CTC TGG ATGA) and/or SEQ ID NO. 13 (P2-Forward GGA GTA TTGGGA CCG GAAC) and SEQ ID NO. 14 (P2-Reverse GTT GTA GTA GCC GCG CAG GT).

In various embodiments wherein when the genetic variant is associatedwith the SNP of the gene encoding CYP2C19 enzyme, said SNP resulting inincompatibility of the pharmaceutical compound Clopidogrel the primerpair comprises SEQ ID NO. 15 (Forward-TGCAATAATTTTCCCACTATCATTG), andSEQ ID NO. 16 (Reverse-CTCCAAAATATCACTTTCCATAAAAGCA),

In various embodiments where the housekeeping gene comprises GAPDH, thekit further comprises the primer pair SEQ ID NO. 17 (GAPDH-ForwardGGAAGGTGAAGGTCGGAGTC CTC) and SEQ ID NO. 18 (GAPDH-ReverseCCTGGAAGATGGTGATGGGATTTC).

In various embodiments the kit further comprises the primer pair SEQ IDNO. 17 (GAPDH-Forward GGAAGGTGAAGGTCGGAGTC CTC) and SEQ ID NO. 18(GAPDH-Reverse CCTGGAAGATGGTGATGGGATTTC) for amplifying a housekeepinggene GAPDH.

The resulting kit may be highly specific and cost-effective screening ofHLA-B*1502 or other genetic variants specific to the morpholinossequences used.

The kit can also comprise one or more solutions, for example ahybridization buffer. The hybridization buffer can for example includebut is not limited to phosphate, citrate, Tris, Hepes(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), and PIPES(Piperazine-1,4-bis(2-ethanesulfonic acid) buffers, hypersolutes (forexample, mannosylglycerate) or any other buffer solution, optionallycontaining, denaturing agents, salts, inert polymers, surfactants, amongothers. The one or more solutions in the kit can also include any one ofthe electrolytes as described herein. The one or more solutions of thekit can be supplied in wells of one or more microplate(s), for example a384-well microplate, or in containers for later application in the wellsof the microplate(s).

By “comprising” it is meant including, but not limited to, whateverfollows the word “comprising”. Thus, use of the term “comprising”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub-generic groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples.

Examples Preparation of the Nanoprobes

Preparation of the nanoprobes used in this study followed a protocolsimilar to that reported previously. Briefly, the MORs modified withdisulfide amide at the 3′ terminal (Gene Tools, LLC) were treated withdithiothreitol to reduce the disulfide bond, and purified by using anNAP-5 column (GE Healthcare). The gold nanoparticles (40 nm-diameter,˜0.1 nM, Ted Pella, Inc.) were mixed with ˜2 □M of thiolated MORs, 10 mMof phosphate buffer (pH 7.5), and allowed to incubate at roomtemperature overnight. Next, the MOR-nanoparticle conjugates were washedfor at least 5 times with a phosphate buffer solution (5 mM, pH 7.5) bycentrifugation to remove the unreacted MORs. The nanoprobes obtainedwere stable for at least 6 months when stored at 4° C. Before use, thenanoprobe solution should be uniformly dispersed by vortex.

gDNA Extraction.

Human gDNA samples can be extracted from whole blood. The extraction canbe performed with the use of the following commercial kit GentraPuregene DNA extraction kit (Qiagen), according to the manufacturer'sinstruction. Quantity (ng/□l) and quality of the gDNA samples can bechecked by absorbance measurements using Nanodrop 1000 (ThermoScientific). The quality of the samples can be characterized by theratio of absorbance at 260 nm and 280 nm (A260/A280 ratio), whichtypically varies from 1.6 to 2.0.

PCR.

The PCR solution with a final volume of 25 □L would typically contain˜10 ng of gDNA, 12.5 □L of 2× master mix (Fermentas or Promega), and 1□M of the forward primer and 100 nM of the reverse primer. PCR cyclingcan be performed on the PTC-200 DNA Engine. The success of the PCR inproducing specific-sized amplicons can be verified by running a 5-□Laliquot of the PCR products on a 1.5% agarose gel stained with SafeView™dye.

T_(m) Measurements.

The synthetic samples or the PCR products were mixed with the specificWT and MUT nanoprobes, respectively, following which, the T_(m) valuesof the target-probe hybrids were measured with the thermal cycler. Thetemperature was increased starting from 32° C. at an interval of 1.0° C.At each temperature, the solution was allowed to incubate for 1 minprior to color visualization. When a clear color change from red tolight grey was observed, the temperature was recorded as T_(m).

Genotype Assignment.

The T_(m) ^(WT)-T_(m) ^(MUT) scatter plots obtained with the syntheticDNA targets (FIG. 2) were used as the standard genotyping diagrams. Theexperimental data point (T_(m) ^(WT), T_(m) ^(MUT)) of samples can beplotted in the diagram, and the genotype is easily determined by theregion where the data point resided.

Detection of DPD Genetic Variants

A dual-nanoprobe genotyping assay gauging the DPD*2A mutation wasdeveloped. Two sets of nanoprobes, i.e., wild-type (WT) and mutant (MUT)probes were developed, by functionalizing gold nanoparticles withmorpholino oligonucleotides (MORs). The oligo sequences are shown inFIG. 1. For the WT nanoprobes, the oligo sequence was perfectly matchedwith the WT DPD gene segment, while for the MUT nanoprobes, the oligosequence was perfectly matched with the mutant allele. The nanoprobeswere stably dispersed in 5 mM of phosphate buffer as a red solution(pH˜8) for at least 6 months. However, the addition of 100 mM of NaClwould lead to irreversible aggregation of the nanoparticles, and thesolution color would turn colorless within 1 min.

The presence of target DNA in solution could increase the stability ofthe nanoprobes due to the increase in surface negative charge of thenanoparticles upon DNA attachment. If the surface density of attachedDNA was high enough, the nanoparticles would be stably dispersed even inthe presence of 100 mM of NaCl. To reveal the thermodynamic property ofthe DNA-nanoprobe hybrids, melting temperature (T_(m)) was measured. AtT_(m), the dissociation of the DNA sequence from the nanoparticlesurface would occur, destabilizing the nanoparticles and resulting insolution color change from red to light grey. The T_(m) data were thenused to determine the genotype of the samples.

To characterize the nanoprobes, T_(m) data were measured in the presenceof 100 mM of NaCl (final concentration) and synthetic DNA samples over abroad concentration range of 5 nM to 500 nM (FIGS. 2 and 5). At roomtemperature (˜25° C.), the WT nanoprobes were stabilized by ≥20 nM ofthe WT targets and could not be stabilized by the MUT targets, while theMUT nanoprobes were stabilized by ≥10 nM of the MUT targets and ≥200 nMof the WT targets. Therefore, the WT and MUT probes exhibited detectionlimits of ˜20 nM and ˜10 nM for the perfectly matched (PM) targets,respectively, while for the single-base-mismatched (1 MM) targets, thesensitivity of both probes was much lower. This might be attributed tothe difference in binding thermodynamics of the target/probe hybrids,which determined the surface density of the target DNA on nanoparticles.The higher binding affinity of the PM targets allowed for more sensitivedetection. In the concentration range of 20 nM to 200 nM, the nanoprobesonly responded to their PM targets. At target concentrations of ≥200 nM,the MUT nanoprobes responded to either the PM or MUT targets. The T_(m)difference induced by a single-base mismatch between the target and theprobe was ˜11° C. For the heterozygote samples, the T_(m) was determinedby the PM concentration.

The data shown in FIG. 5 could be presented as a T_(m) ^(WT)-T_(m)^(MUT) scatter plot, which could serve as the standard genotypingdiagram (FIG. 6). The diagram was divided into three regions accordingto the specific sequence type of the samples. For an unknown sample,once the data of T_(m),WT and T_(m),MUT were obtained, the genotypecould be assigned based on the region where the data point lay in thestandard genotyping diagram.

For human genomic DNA (gDNA) samples, PCR amplification can be conductedto produce sufficient amount of the specific target sequence of the DPDgene since the detection sensitivity of the assay was ˜10-20 nM. FIGS. 3and 4 show the PCR primers and thermal cycling parameters. FollowingPCR, two aliquots of the PCR products can be directly mixed with WT andMUT nanoprobes, respectively, then Tm values of the hybrids of WTprobe/amplicon and MUT probe/amplicon can be measured. The obtained datapoint (T_(m)WT, T_(m)MUT) can be plotted in the standard genotypingdiagram to determine the sample's genotype.

In summary, the dual-nanoparticle assay kit to gauge DPD*2A genotypeonly required a standard thermal cycler, which allowed forcost-effective detection. The highly specific plasmonic nanoprobesensured the accurate genotyping based on colorimetric signals.

Detection of HLA-B Genetic Variants

Five signature sequence motifs of HLA-B*1502 exons 2 and 3 have beenused to identify the specific allele, as shown in FIG. 7. The presenceof the five sequence motifs on a same DNA molecule indicates the carrierstate of HLA-B*1502. The specificity of the method is relatively high.The alleles with across reactions with HLA-B*1502 include B*1588,B*15112, B*15121, B*15144, B*15170, B*15194, B*15213, B*15214 andB*15223. These alleles are all very rare with no allele frequencyreported in the world populations.

Two separate asymmetric PCR (aPCR) reactions were used to detect theHLA-B*1502 allele on the basis of the specificity of either the primersor the nanoprobes (FIG. 8). In the first aPCR reaction (PCR1), theforward primer (Primer F1) is specific to the sequence motif 1 and thereverse primer (Primer R1) is specific to the motif 5, while the motifs2-4 are gauged by Probes 1-3, respectively. The amplicon size is 429 nt,covering part of exon 2, entire intron 2, and part of exon 3. Thepositive results of all the three nanoprobe assays indicate the presenceof the five signature motifs, but with combination ambiguities as shownin FIG. 9. Therefore, a second PCR reaction (PCR2) is employed toachieve an unambiguous determination. In PCR2, the forward primer(Primer F2) is specific to motif 2, and the reverse primer (Primer R2)is specific to motif 4, while motif 3 is gauged by Probe 2. The positiveresult of the nanoprobe assay ensures the same phase of the threesequence motifs, ruling out the ambiguities shown in FIG. 3. An internalcontrol (gauging the GAPDH gene) is included in PCR2 to monitor thevalidity of the PCR reactions.

The morpholino oligonucleotides (MORs) modified with disulfide amide atthe 3′ terminal, are shown in FIG. 10

Behavior of the Nanoprobes

Nanoprobes were used to gauge three sequence motifs in this study. Theability of the nanoprobes to discriminate between the perfectly matched(PM) targets and the mismatched (MM) targets was examined by usingsynthetic oligonucleotide samples (FIG. 11). On the basis of thedatabase, we selected sequences most similar to HLA-B*1502 signaturemotifs as mismatched (MM) samples. As shown in FIG. 4, most of thesequences could not generate signals over a wide concentration range (upto 500 nM) at temperatures higher than 32° C. For nanoprobe 2,single-base mismatches needed to be distinguished. FIG. 4 shows that thesingle-base mismatches generally reduced the T_(m) by 8-10° C., allowingfor allele discrimination

HLA-B*1502 Genotype Determination

Aliquots of the PCR1 products (ratio 1:1) were mixed with probes 1, 2and 3 in three microtubes, respectively. Aliquots of the PCR2 products(ratio 1:1) were mixed with probe 2 and GAPDH probe in 2 microtubes,respectively. The mixed solutions were incubated at 40° C. for 5 min. Ifthe GAPDH probe solution is red in color, the PCR reactions are valid.In this case, when all the other 4 solutions are red in color, thesample is HLA-B*1502 positive, otherwise, the sample is HLA-B*1502negative. If the GAPDH probe solution becomes colorless, the assay isinvalid and needs to be repeated. The assay workflow is shown in FIG.12.

The nanoprobes were designed to be highly specific in recognition ofnucleic acid targets, and generate colorimetric signals that can beeasily visualized. This technology allows for accurate end-pointdetection with a simple workflow and standard equipment. On the basis ofthis platform, a cost-effective HLA-B*1502 screening kit was developed.

A simple nanoprobe-based assay has been developed for the HLA-B*1502allele determination. The method shows unique features and superiorperformance as compared to other available techniques. Firstly, theassay is highly specific, ensuring accurate detection. Due to the largenumber of HLA-B alleles that are very close in sequence to theHLA-B*1502 allele and the high frequency of phase ambiguity, falsepositive commonly occurs in the screening of HLA-B*1502 carrier.Secondly, the nanoprobe technique allows for distinct colorimetricdetection, greatly reducing the screening cost. The high specificity ofthe method is very important as it makes it accurate and cost effective.Analysis of the cost-effectiveness of HLA-B*1502 screening beforecarbamazepine treatment indicates that the genetic testing cost iscritical, especially for the populations with low and medium prevalenceof HLA-B*1502 allele. The visualizable signals simplify the detection,and exclude the requirement of expensive detectors. As a result, thenanoprobe-based genetic testing provides a specific and less costlyassay for the screening of HLA-B*1502.

Detection of CYP2C19 Genetic Variants

Two sets of nanoprobes, i.e., wild-type (WT) and mutant (MUT) probes,were prepared by functionalizing gold nanoparticles with morpholinooligonucleotides (MORs). The MOR oligo sequences are shown in FIG. 16.The WT nanoprobe sequence is perfectly matched with the WT CYP2C19 genesegment, while the MUT nanoprobe sequence is perfectly matched with theCYP2C19*2 mutant allele. The as-prepared nanoprobes were red in colorand stable for at least 6 months. However, irreversible aggregation ofthe nanoparticles could be induced by the addition of 500 mM of NaCl,causing the solution to change from red to light grey within 2 min.

When target DNA sequences were added in the nanoprobe solution, thenanoprobes became more stable even in the presence of 500 mM of NaCl,because DNA attachment increased the surface negative charge of thenanoparticles. As solution temperature was raised, the DNA-nanoprobehybrids would be dissociated at the melting temperature (T_(m)), causingthe nanoparticles to become unstable and changing the solution colorfrom red to light grey. The value of T_(m) could be obtained byvisualizing the solution color change. The T_(m) data were then used todetermine the genotype of the samples.

FIG. 17 shows the nanoprobe behavior in response to synthetic DNAsequences (see FIG. 18). At room temperature (˜25° C.), both of the WTand MUT nanoprobes were stabilized by ≥10 nM of the targets. The T_(m)difference induced by a single-base mismatch between the target and theprobe was ˜5-7° C. For the heterozygote samples, the T_(m) wasdetermined by the perfectly matched target concentration.

FIG. 19 shows the data as a T_(m) ^(WT)-T_(m) ^(MUT) scatter plot, whichcould serve as the standard genotyping diagram. Three linear regions inthe diagram are corresponding to the three genotypes, i.e. *1/*1, *1/*2and *2/*2. For an unknown sample, once the data of T_(m) ^(WT) and T_(m)^(MUT) were obtained, the genotype could be assigned based on the regionwhere the data point lied in the standard genotyping diagram.

To test genomic DNA (gDNA) samples, PCR amplification of the genesequence segments flanking the single nucleotide polymorphism (SNP) canbe conducted by using primers shown in FIG. 20. The PCR thermal cyclingparameters are shown in FIG. 21. Following PCR, two aliquots of the PCRproducts are mixed with the WT and MUT nanoprobes, respectively, thenT_(m) values of the hybrids of WT probe/amplicon and MUT probe/ampliconcan be measured. The obtained data point (T_(m) ^(WT), T_(m) ^(MUT)) canbe plotted in the standard genotyping diagram to determine the sample'sgenotype.

In summary, a dual-nanoparticle assay kit to gauge CYP2C19*2 genotypewas developed. The assay is cost-effective and accurate, and would beapplicable for point-of-care testing.

The invention claimed is:
 1. A method comprising: providing a firstnano-particle coupled to at least one synthetic morpholino nucleic acidprobe comprising a target-complementary region comprising a basesequence that is a perfect match to a genetic variant; combining asample containing a nucleic acid suspected of containing the geneticvariant and the first nano-particle to provide a first mixture underconditions that allow hybridization of the synthetic morpholino nucleicacid probe and the nucleic acid suspected of containing the geneticvariant; sequentially heating the first mixture and determining amelting temperature of a hybridization complex between the syntheticmorpholino nucleic acid probe and the nucleic acid suspected ofcontaining the genetic variant; comparing the melting temperaturedetermined for the hybridization complex with a standard value todetermine whether the sample comprises nucleic acid containing thegenetic variant; and detecting the genetic variant, wherein the geneticvariant is associated with one or more of: a single nucleotidepolymorphism of a gene encoding a dihydropyrimidine dehydrogenase (DPD)enzyme; a single nucleotide polymorphism of a gene encoding a CYP2C19enzyme; an allelic variation of a gene encoding a human leukocyteantigen; or combinations thereof.
 2. The method according to claim 1,wherein the standard value is established by: determining a meltingtemperature of a complex of the first nano-particle coupled to at leastone synthetic morpholino nucleic acid probe comprising atarget-complementary region comprising a base sequence that is a perfectmatch to the genetic variant and the corresponding wildtype nucleicacid; or determining a melting temperature of a complex of a secondnano-particle coupled to at least one synthetic morpholino nucleic acidprobe comprising a target-complementary region comprising a basesequence that is a perfect match to the corresponding wild type basesequence.
 3. The method according to claim 1, wherein the method furthercomprises: providing a second nano-particle coupled to at least onesynthetic morpholino nucleic acid probe comprising atarget-complementary region base sequence that is a perfect match to thecorresponding wild type base sequence of the genetic variant; combininga sample with the second nano-particle to provide a second mixture underconditions that allow hybridization of the synthetic morpholino nucleicacid probe and the nucleic acid suspected of containing the geneticvariant; and sequentially heating the second mixture and determining themelting temperature of the hybridization complex between the syntheticmorpholino nucleic acid probe and the nucleic acid suspected ofcontaining the genetic variant; comparing the melting temperaturedetermined for the hybridization complex comprising thetarget-complementary region comprising a base sequence that is a perfectmatch to the genetic variant with the melting temperature determined forthe hybridization complex comprising the target-complementary regionbase sequence that is a perfect match to the wild type; and detectingthe genetic variant when the melting temperature of the hybridizationcomplex comprising the target-complementary region comprising a basesequence that is a perfect match to the genetic variant is higher thanthe melting temperature determined for the hybridization complexcomprising the target-complementary region base sequence that is aperfect match to the wild type.
 4. The method according to claim 1,wherein the melting temperature is indicated by a color change of themixture upon dissociation of the probe and the sample nucleic acid. 5.The method according to claim 1, wherein the synthetic morpholinonucleic acid probe comprises a target-complementary region being fullycomplementary to a genetic variant of a gene encoding dihydropyrimidinedehydrogenase (DPD) and having the base sequence set forth in SEQ IDNO:1 and the wild type synthetic morpholino nucleic acid probe comprisesa target-complementary region being fully complementary to the wild typesequence having the base sequence set forth in SEQ ID NO:2 or whereinCYP2C19 enzyme morpholino nucleic acid probe comprises atarget-complementary region being fully complementary to a sequencerelated to a genetic variant of a gene encoding CYP2C19 enzyme havingthe base sequence set forth SEQ ID NO:7 and the wild type syntheticmorpholino nucleic acid probe comprises a target-complementary regionbeing fully complementary to the wild type sequence having the basesequence set forth in SEQ ID NO:8.
 6. The method according to claim 1,wherein the morpholino nucleic acid probe optionally comprises atarget-complementary region being fully complementary to an allelicvariant of the gene encoding human leukocyte antigen having the basesequence set forth in SEQ ID NO:3; SEQ ID NO:4; and SEQ ID NO:5.
 7. Themethod according to claim 6, further comprising a nano-particle coupledto a morpholino nucleic acid probe comprising a target-complementaryregion that is fully complementary to a housekeeping gene.
 8. The methodaccording to claim 7, wherein the housekeeping gene is GADPH having amorpholino sequence set forth in SEQ ID NO:6.
 9. The method according toclaim 1, further comprise amplifying the nucleic acid suspected ofcontaining the genetic variant prior to mixing the nucleic acid with thefirst nano-particle or a second nano-particle.
 10. The method accordingto claim 9, wherein when the genetic variant is associated with an SNPof the gene encoding dihydropyrimidine dehydrogenase (DPD), the nucleicacid is amplified using a primer pair comprising SEQ ID NO:9 and SEQ IDNO: 10, or wherein when the genetic variant is associated with theallelic variation of the gene encoding human leukocyte antigen, thenucleic acid is amplified with a primer pair comprising SEQ ID NO:11 andSEQ ID NO:12 and/or SEQ ID NO:13 and SEQ ID NO:14, or wherein when thegenetic variant is associated with an SNP of the gene encoding CYP2C19enzyme, the nucleic acid is amplified with a primer pair comprising SEQID NO:15, and SEQ ID NO:16, optionally further comprising using primerpair SEQ ID NO:17 and SEQ ID NO:18 for amplifying a housekeeping gene.11. The method according to claim 1, wherein the nanoparticle comprisesa metal nanoparticle or a colloidal metal, the metal optionallycomprising a noble metal.
 12. The method according to claim 1, whereinthe nanoparticle comprises a semiconductor or magnetic colloidalmaterials, or wherein the synthetic morpholino nucleic acid probe iscovalently coupled to the nanoparticle.